Influenza is a global infectious viral disease of great public health concern affecting millions of people every year. Influenza viruses cause increased morbidity and mortality in the range of 20,000-40,000 death/yr in USA alone and economic loss of 10-20 billions of dollars/yr. Vaccines are in short supply essentially every year. Furthermore, there is a bird flu or avian flu virus posing a global pandemic threat against which there is no effective vaccine and massive amount of vaccine will be required to combat the pandemic if and when it occurs. With the increasing aging population, this risk is increasing every year and a possibility of a new pandemic looming. Therefore a highly attenuated live vaccine is urgently needed. Our preliminary studies have identified that in A/WSN/33(H1N1) virus, a conserved CCHH motif (the putative zinc finger motif) of influenza virus matrix protein (M1) can be mutated without affecting virus replication in MDCK cells in culture. Some CCHH mutant viruses were highly attenuated in mice and protected mice against lethal WT virus challenge. In this project, we want to create these mutations in A/PR8/34(H1N1) virus which is commonly used for making influenza vaccine and test the effect of these mutations in mice virulence, growth in cell cultures and embryonated chicken eggs and protection efficiency against WT lethal virus challenge. In addition, other mutations in other genes will be added to CCHH mutations and their effect on virulence, stability, growth in cell cultures and embryonated eggs and protection efficiency will be determined. A highly attenuated and avirulent PR8 virus can be used as master strain for generating highly attenuated live influenza vaccine. Live attenuated vaccine produced in this way will be less thermo-labile than the presently used cold adapted vaccine. Live vaccine can be grown easily in cell culture or in embryonated chicken eggs. A live vaccine can be delivered easily and a smaller dose will be required to immunize people providing plenty supply of vaccine. Live vaccine will also produce broader and long lasting protective immunity against epidemic and/or pandemic viruses. PUBLIC HEALTH RELEVANCE: Influenza is a global infectious viral disease of great public health concern affecting millions of people every year. The goal of this project is to create highly attenuated PR8 viruses which can be used as master strains for generating live attenuated influenza vaccine. Live attenuated vaccines can be produced in mass quantity, delivered easily and will produce broader and long lasting protective immunity against virulent influenza viruses.